The Epstein-Barr virus (EBV) is an extremely common environmental agent infecting 80-100 percent of the individuals around the world. It is the causative agent of infectious mononucleosis (IM) in humans, EBV has also been implicated in the pathogenesis of Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), and B lymphocyte neoplasms arising in immunosuppressed patients. Circumstantial evidence has also indicated a possible role for this virus in human autoimmune disease such as rheumatoid arthritis and Sjogren's Syndrome.
The initial or primary EBV infection may be acute or sub-clinical. Acute viral infection leads to the production of specific nuclear antigens (termed EBNA-I and EBNA-II), an "early antigen" (EA) complex, viral capsid antigens (VCA), and other virus-associated molecules. This is followed by a long period during which the EBV infection is latent in B lymphocytes present in the circulating blood, lymph nodes, spleen and salivary glands.
A latent infection is one in which a virus is present intracellularly in an unexpressed or partially expressed state. Latent viral infections can be reactivated. Although the host factors that control latency in vivo are poorly understood, there is some evidence to suggest that failure of one or more immune mechanisms is an important factor.
The serological and cell-mediated immune responses that follow primary infection by EBV are well documented and reflect the host's response to the viral immunogenic determinants expressed during the course of infection. In the context of native viral proteins, immunogenic determinants are those parts of a protein that elicit the antibody response when the whole, native protein is used as immunogen. These immunogenic determinants are believed to be confined to a few loci on the molecule.
On the other hand, a region of a protein molecule to which an antibody can bind is defined as an antigenic determinant. The detection of viral antigenic determinants in tissues as well as the profile of the patient's response to viral immunogenic determinants are becoming increasingly useful in the diagnosis of EBV-associated diseases.
The EA complex is of particular interest since antibodies to this complex are frequently present in high titers in patients with EBV-associated diseases as opposed to latently infected but non-diseased control populations. That is, humans acutely infected with Epstein-Barr virus (EBV) develop antibodies against a diffuse early antigen (EA-D). Subsequently, anti-EA-D antibodies disappear as the virus enters a phase of latency and do not reappear unless the virus is reactivated.
The EA complex is now known to consist of two distinct protein antigens designated diffuse (D) and restricted (R) based on the distribution of immunofluorescent staining in EBV-infected cells. Antibody to EA-D causes diffuse staining of the nucleus and cytoplasm in both acetone- and methanol-fixed cells. In contrast, EA-R staining is restricted to the cytoplasm in acetone-fixed cells and is not present in methanol-fixed cells.
The anti-EA activity of sera of patients with IM and NPC is directed primarily against EA-D, whereas immunoreactivity in sera of patients with BL is directed mainly against EA-R. In addition, antibodies to the EA complex are of importance in patients with EBV-associated malignancies since antibody titers tend to vary with disease course.
Thus, assays for the presence of both EA-D and anti-EA-D antibodies are of importance in several common clinical situations.
Anti-EA-D antibodies have heretofore been assayed using EA-D antigen obtained from EBV-infected cells. The immunofluorescent technique of Henle et al., Science, 169, 188-190 (1970) uses whole-cell preparations without any antigen purification. However, the use of such crude preparations results in false positive results for patients whose serum also contains antibodies to mammalian nuclear and cytoplasmic antigens.
More recently, Luka et al., J. Immunol. Meth., 67, 145-156 (1984), reported developing an enzyme-linked immunosorbent assay (ELISA) for anti-EA-D antibodies using EA-D target antigen purified from EBV-infected cells by immunoaffinity chromatography. While the method of Luka et al. diminishes the false-positive problem associated with the use of whole cell preparations, it still requires production and handling of infectious materials.
Accordingly, it would be desirable to develop improved reagents and methods for assaying for the presence of EA-D and anti-EA-D antibodies in a body sample so as to allow diagnosis of EBV involvement in disease, as well as diagnosis of the stage of a disease such as infectious mononucleosis (IM), and limit or avoid handling of infected cell cultures.
Recent studies have shown that chemically synthesized polypeptides corresponding to short linear segments of a protein's primary amino acid residue sequence can be used to induce antibodies that immunoreact with the native protein, Lerner et al., Nature, 299, 592 (1982) and Sutcliffe et al., Science, 219, 260 (1983). In addition, some studies have shown that synthetic polypeptides can immunoreact with antibodies induced by native proteins. Rhodes et al., J. Immunol. 134, 211 (1985). Thus, some synthetic polypeptides can immunologically mimic the immunogenic and antigenic determinants of native proteins.
However, as is well known in the art, the application of synthetic peptide technology still suffers several shortcomings. For instance, the identification of peptides capable of mimicking antigenic determinants on a native protein requires knowing, inter alia, the amino acid residue sequence of the protein. Whereas the amino acid residue sequence can be predicted from the nucleic acid sequence of the gene coding for the protein, such a prediction can only be made if the correct reading frame of the gene is known.
The nucleic acid sequence of the EBV genome has been known since publication of the Baer et al., Nature, 310, 207 (1984) article. However, neither the EA-D protein gene nor its reading frame has heretofore been identified with the viral genome. Furthermore, even if a protein's amino acid residue sequence is known, methods for identifying the loci in the protein that constitute the immunogenic and antigenic determinants are experimental in nature and do not yield predictable results. There are at least two reasons for this. First, without knowing a protein's three-dimensional structure there is no reliable method for determining which linear segments of the protein are accessible to the host's immune system. Second, whether the three-dimensional structure is known or not, short linear polypeptides often appear not to have the ability to mimic the required secondary and tertiary conformational structures to constitute appropriate immunogenic and antigenic determinants, Tainer et al., Nature, 312, 127 (1984).